Identification of inhibitors of heparin-growth factor interactions from combinatorial libraries of four-component condensation reactions.

Chemical libraries based on four-component condensation (4CC) reactions of isocyanides were constructed to identify compounds capable of blocking heparin binding to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The reaction products in the synthesized libraries contain heparin mimetic functional groups such as carbohydrates, sulfonates, carboxylates, and hydroxy groups. These libraries have been screened for the inhibition of heparin binding to growth factors such as VEGF and bFGF. Single point screening at 5.0 microM of the 18,720 reaction products generated 26 candidates. The IC50S of these 26 compounds were determined using HPLC-purified products and 20 of the 26 showed significant inhibition of heparin binding to VEGF and/or bFGF. Eighteen of the 20 confirmed active compounds have a linear extended structure. Structures identified in this library revealed an initial relationship of structure and activity, thus providing direction for further investigation of this type of heparin mimetic libraries.

Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-1.

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.

A21978C, a complex of new acidic peptide antibiotics: isolation, chemistry, and mass spectral structure elucidation.

A21978C, produced by Streptomyces roseosporus, NRRL 11379, is a complex of new acidic lipopeptolide antibiotics which inhibits Gram-positive bacteria. HPLC separation of the various components from the purified complex resulted in the isolation of A21978C1, -C2 and -C3 (major components) and -C4, -C5, and -C0 (minor components). Each of these components was fermented with cultures of Actinoplanes utahensis (NRRL 12052) to give the identical inactive peptide ("A21978C nucleus") by removal of the fatty acid acyl groups from the N-terminus. This peptide was composed of 13 amino acids: L-kynurenine, L-threo-3-methylglutamic acid, L-asparagine, L-aspartic acid (3 residues), glycine (2 residues), L-tryptophan, L-ornithine, D-alanine, D-serine and L-threonine. The amino acid sequence was determined using a combination of the Edman degradation and gas chromatography mass spectrum (GC-MS) analysis of appropriately derivatized peptides obtained from partial hydrolysis. Each major component was shown to be acylated with a branched chain fatty acid at the N-terminus and the structure of this fatty acid was determined by 1H NMR and mass spectral methods. A structure for A21978C was assigned on the basis of this degradative and physico-chemical information.

Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography.

T4 RNA ligase has been used to construct a series of defined oligoribonucleotides. Hexamer or pentamer blocks were synthesized first by multiple additions of mononucleotide diphosphates to trimers with T4 RNA ligase and removal of the terminal phosphate with alkaline phosphatase; inhibitors of the ligase were removed by passing the sample over a 1-ml reverse-phase octadecasilyl column. The two nucleotide blocks were then ligated to give undecamers. Yields for the individual ligations ranged from 85 to 100% for acceptors lacking uridines and at least 70% for those containing uridines. The overall yield of the undecamer relative to the starting trimers was about 10%. Each round of ligation averaged about 8 h; the time required to synthesize each undecamer was 1 to 2 weeks. Optimization of the steps to achieve this is described in detail.

Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

A revised primary structure for neocarzinostatin based on fast atom bombardment and gas chromatographic-mass spectrometry.

The amino acid sequence of the antitumor protein neocarzinostatin was revised on the basis of mass spectrometric studies. Gas chromatographic mass spectrometry on the O-trimethylsilyl polyaminoalcohol derivatives of peptide mixtures derived from tetra S-carboxymethyl-neocarzinostatin were used to partially sequence neocarzinostatin. In addition, fast atom bombardment-mass spectrometric experiments on neocarzinostatin and its tryptic fragments gave the molecular weights of various peptides and, in some cases, partial sequence information. The revised sequence involved reordering of two chymotryptic peptides, the identification of a new di- and tripeptide sequence (Ala-Asp and Ala-Ser-Thr), the repositioning of Trp at position 39, and the assignment of the remaining Asx residues. The revised structure for neocarzinostatin (Mr = 11,105) now shows considerable homology with the other antitumor antibiotic proteins macromomycin and actinoxanthin.

A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing.

Complete amino acid sequences have been established for 19 muscle-related proteins and these proteins are each sufficiently abundant to suggest that their mRNA levels are about 0.4% or higher. Based on these considerations, a simple theoretical analysis shows that clones for most of these proteins can be identified within a complementary DNA library by sequencing cDNA inserts from 150-200 randomly selected clones. This procedure should not only rigorously identify specific clones, but it could also uncover amino acid sequence variants of major muscle proteins such as the troponins. We have determined sequences for about 20,000 nucleotides within 178 randomly selected clones of a rabbit muscle cDNA library, and report here that in addition to finding sequences encoding the two known skeletal muscle isotypes of troponin C, we have discovered sequences encoding two forms of troponin T. Over the region of nucleotide sequence overlap in the troponin T clones, the new isotype diverges significantly from its counterpart. Altogether, clones for 13 of the 19 known muscle-specific proteins were identified, in addition to the clone for the new troponin T isotype.

Primary structure of macromomycin, an antitumor antibiotic protein.

The antitumor protein macromomycin is a single chain polypeptide of 112 amino acid residues cross-linked by two intramolecular disulfide bonds. The protein was reduced and S-alkylated with 2-mercaptoethanol in 8 M urea followed by treatment with iodoacetic acid. Tryptic digestion of tetra-S-carboxymethyl macromomycin gave four tryptic peptides which were fractionated by gel permeation on Sephadex G-50. The amino acid sequence of the tryptic peptides and the overlap sequences were determined by a combination of automated Edman degradation analysis, gas chromatographic mass spectrometry, and fast atom bombardment mass spectrometry. A comparison of the structures of macromomycin, actinoxanthin, and neocarzinostatin suggests that they belong to a family of related proteins.

Diabetic nephropathy associated with fibrin formation.

This communication describes the electron microscopic study of a renal biopsy specimen from a patients with diabetic nephropathy, the nephrotic syndrome, and renal insufficiency. There were large amounts of electron dense materials within glomerular basement membranes and masses of fibrin within glomerular capillaries and Bowman's spaces. The presence of glomerular fibrin suggests that thrombosis may be pathogenetically related to diabetic nephropathy.

Resonance Raman spectroscopy of specifically [epsilon-15N]lysine-labeled bacteriorhodopsin.

The possible interaction of a second lysine with the retinylidene Schiff base of bacteriorhodopsin (Lewis, A., Marcus, M. A., Ehrenberg, B. & Crespi, H. (1978) Proc. Natl. Acad. Sci. USA 75, 4642-4646) has been investigated by specific incorporation of 15N into the epsilon-amino groups of the lysine residues. Comparison of resonance Raman spectra of bacteriorhodopsin grown on 100%, 0%, and 50% labeled lysine demonstrates that 15N isotope effects on the Schiff base vibration can be accounted for by 15N labeling only at the Schiff base nitrogen. Our data also provide in situ confirmation of the linkage of the retinal chromophore with the epsilon-amino nitrogen of lysine.

Advances in gas chromatographic mass spectrometric protein sequencing. 2--Application to membrane proteins.

The primary structure of the integral membrane protein bacteriorhodopsin was determined by an efficient combination of gas chromatographic mass spectrometric techniques with the Edman degradation. This combination of methodologies circumvented many of the experimental difficulties associated with the insolubility of bacteriorhodopsin and its primary degradation fragments in aqueous buffers. Specifically, in the gas chromatographic mass spectrometric analysis of the cyanogen bromide peptides derived from bacteriorhodopsin, it has been possible to identify homoserine-containing peptides which served as a starting point for the construction of C-terminal sequences. In most cases this C-terminal sequence constructed from the gas chromatographic mass spectrometric peptides overlapped the N-terminal sequence derived in an Edman degradation experiment, thereby completing the structure of the fragment. Furthermore, the specific identification of methionine-containing peptides required to establish the order of the cyanogen bromide fragments was accomplished by direct analysis of the complex mixtures generated by partial hydrolysis of segments of the protein. These data made it possible to determine the sequence of a large portion of bacteriorhodopsin solely from cyanogen bromide cleavage, one of the few specific reactions compatible with the solubility properties of this hydrophobic protein. Finally, the gas chromatographic mass spectrometric sequence data have been used to assign or confirm amino acids where the Edman data was ambiguous. These gas chromatographic mass spectrometric techniques resulted in an efficient and reliable determination of the complete sequence of this membrane protein which is 248 amino acids long.

Mass spectra of partial protein hydrolysates as a multiple phase check for long polypeptides deduced from DNA sequences: NH2-terminal segment of alanine tRNA synthetase.

A strategy has been developed for rapid and accurate determination of the amino acid sequence of large proteins, such as many of the members of the class of proteins known as aminoacyl tRNA synthetases. This strategy involves combining DNA sequencing of the gene for the protein of interest with gas chromatographic mass spectrometric identification of tetra- and pentapeptides in partial hydrolysates of the entire protein or very large fragments thereof. These peptides are matched to blocks of codons at locations scattered throughout the entire structural gene. Tetra- and pentapeptide sequences are sufficiently long that they are unlikely to be repeated in the protein sequence or to occur in an incorrect reading frame; therefore, they can be placed at unique clusters of codons on the DNA. This procedure rigorously establishes the proper phasing of the DNA throughout the entire length of the structural gene, and the protein sequence is thereby accurately read from the DNA sequence. This approach is being used to determine the amino acid sequence of EScherichia coli alanine tRNA synthetase, a protein that has approximately 900 amino acids. This paper reports the sequence of the first 165 amino acids from the NH2 terminus.

Amino acid sequence of bacteriorhodopsin.

The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.

Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins.

The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y.A., Abdulaey, N.G., Feigina, M.Y., Kiselev, A.V. & Lobanov, N.A. (1977) FEBS Lett. 84, 1-4]. Chymotryptic cleavage of bacteriorhodopsin produced two fragments, C-1 (Mr 19,000) and C-2 (Mr 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.